The formation of biofilm communities poses a major challenge to the prevention and treatment of infectious diseases. It is well established that bacteria within biofilm communities are highly recalcitrant to elimination with biocides, classical antibiotics and disinfectants. Biofilm formation has been suggested to enhance the survival and persistence of numerous pathogenic bacteria in their natural reservoirs. Furthermore, treatment of biofilm-related infections require long-term antibiotic therapies that favor the acquisition of resistance mechanisms while the high cell density in the biofilm environment provides an ideal scenario for horizontal gene transfer. Small molecules capable of inhibiting biofilm formation can provide new means to prevent and treat infectious disease. Vibrio cholerae of serogroups O1 and O139 causes the diarrheal disease cholera, while the closely related species Vibrio vulnificus can cause fatal systemic food and waterborne infections. Both species have been shown to produce quorum sensing regulated biofilms and possess analogous signal transduction pathway responding to autoinducer 2. Recently, transition state analogs of the enzyme methylthioadenosine nucleosidase (MTAN or pfs nucleosidase) that block the biosynthesis of autoinducer 2 were found to inhibit biofilm formation in V. cholerae. Transition state analogs are compounds with a chemical structure that resembles the "high energy" intermediate to which a substrate is converted in a catalytic reaction but to not undergo further conversion to products and act as potent inhibitors. In this application we will use the transition state inhibitors of MTAN MT- immucillin-A and MT-DADMe-immucillin-A as a tool to improve our understanding of the role of MTAN and autoinducer 2 in quorum sensing and biofilm formation in V. cholerae and V. vulnificus. In Aim 1 we will determine the mechanism by which inhibition of MTAN diminishes biofilm formation in V. cholerae. To this end, we will combine chemical inhibition studies, quorum sensing mutants and the manipulation/measurement of the intracellular concentration of second messengers and key metabolites to explain the effect of these inhibitors on biofilm formation. Quorum sensing co-regulates biofilm formation and the expression of virulence factors in pathogenic Vibrios. Therefore, in Aim 2 we will determine if transition state inhibitors of MTAN act through the same pathway in V. cholerae and V. vulnificus and study their effect on the expression of virulence factors in the two pathogenic Vibrios. Again, we will combine chemical inhibition studies, microbial genetics and in vitro assays of virulence to establish the role of MTAN in the expression of virulence factors. PUBLIC HEALTH RELEVANCE: The formation of bacterial biofilms poses a major challenge to the prevention and treatment of infectious diseases since bacteria within biofilm communities are highly recalcitrant to elimination with biocides, classical antibiotics and disinfectants. A better understanding of the environmental signals and response pathways leading to biofilm formation could facilitate the rational design of biofilm inhibitors to prevent and treat infectious disease. In this application we propose to use a combined chemical biology and microbial genetic approach to increase our understanding of the mechanism that triggers biofilm formation in two pathogenic Vibrios (Vibrio cholerae and Vibrio vulnificus) responsible for major food and waterborne illness.